We have been working on the functional inter-relationships between cytoskeletal dynamics and ion channel functions since I joined the NIA. I previously provided evidence that actin filaments regulate neuronal calcium homeostasis and showed that, by stabilizing calcium homeostasis, actin filament depolymerization is neuroprotective (1). By performing calcium imaging studies using agents that depolymerize (colchicine) or stabilize (taxol) microtubules, I provided evidence that microtubule dynamics have an impact on neuronal calcium homeostasis. In 2002 we reported that Ca2+ release from intracellular organelle such as endoplasmic reticulum and mitochondria through inositol tris-phosphate (IP3) and ryanodine mediated pathways is significantly inhibited by actin depolymerization in rat cultured hippocampal neurons (2). [unreadable] Microtubule-associated protein, tau, is the major component of paired helical filaments (PHFs), which form neurofibrillary tangles (NFTs) within degenerating neurons. Frontotemporal dementia and parkinsonism related to chromosome 17 (FTDP-17) is an autosomal dominant hereditary neurodegenerative disorder characterized by personality changes and cognitive dysfunction; mutations in the tau cause FTDP-17 (2). Although several functional changes of tau protein by its genetic mutations are proposed, exact molecular and cellular pathomechanism, which tau mutations cause neurodegeneration, was not known. In order to determine how tau mutations in FTDP-17 alter functions of tau protein and lead to neuronal cell death, we employed SH-SY5Y human neuroblastoma cell lines, which are stably transfected with wild type or mutant tau genes, and studied how ion homeostasis and cell vulnerability are modulated by tau mutations. Two adverse consequences of the tau mutations have been previously proposed: 1) Mutations adjacent to exon 10 (which encodes one of four microtubule-binding domains) alter RNA splicing resulting in an altered ratio of 4-repeat/3-repeat tau(4), and 2) Mutations in coding sequences of the tau gene alter the affinity and capacity of tau to bind microtubules (5). It has been suggested that perturbed cellular calcium homeostasis plays a prominent role in the pathogenesis of Alzheimer's disease, and it was reported that microtubule dynamics can affect cellular calcium homeostasis. We therefore designed experiments to test the hypothesis that tau mutations perturb neuronal calcium regulation (6). Lines of human SH-SY5Y cells stably overexpressing the wild-type tau, the N279K tau mutation or the V337M mutation were produced. The N279K mutation increases expression of 4-repeat tau, but no apparent effect on microtubule binding, while the V337M mutation reduces microtubule binding. The basal [Ca2+]i was essentially identical in cells overexpressing wild-type and V337M or N279K forms of tau. However, cells expressing the V337M tau mutation exhibited a much greater increase of [Ca2+]i in response to depolarization compared to cells overexpressing wild-type or N279K mutant forms of tau. To determine whether enhanced microtubule depolymerization caused by V337M tau was involved in the enhanced [Ca2+]i response to depolarization, we treated cells with taxol prior to depolarization. Taxol completely abrogated the enhanced [Ca2+]i response to depolarization in cells expressing V337M tau, indicating a requirement for microtubule depolymerization in the mechanism whereby the V337M mutation perturbs cellular calcium homeostasis. Depolarization induces calcium influx through voltage-dependent calcium channels (VDCC), and excessive influx through such channels can promote neuronal degeneration. We recorded calcium currents (I-Ca) in cells expressing wild-type or mutant forms of tau. The current density of VDCC was significantly greater in cells expressing V337M tau than in cells expressing wild-type tau or N279K mutant tau. The V337M tau mutation did not affect the current-voltage relationships. Repeated recording of I-Ca at20 s intervals using a brief 30 ms depolarizing pulse revealed that cells expressing wild-type or N279K mutant forms of tau exhibited a progressive run-down of the I-Ca amplitude. In contrast, there was a striking delay in the rundown of I-Ca in cells expressing V337M tau. When cells expressing V337M tau were treated with taxol, the I-Ca rundown was restored to its normal rate. These findings show that the V337M tau mutation enhances calcium influx through VDCC by reducing current rundown by a mechanism requiring microtubule depolymerization. We next isolated L-type, N-type, and other types of VDCC currents using nifedipine to selectively block L-type channels and omega-conotoxin-GVIA to block N-type channels. The amplitudes of I-Ca inhibited by individual VDCC blockers were plotted. The L-type I-Ca in the V337M transfected cells showed the smallest rundown at 400 s compared to N-type and other types of VDCC, indicating that the L-type channel is the most strongly affected by the tau mutation. If enhancement of calcium influx is critical for the pathogenic action of the V337M mutation in FTDP-17, then blocking calcium influx should counteract the neurodegenerative effects of the mutant tau. Cells expressing the V337M tau mutation exhibited an increase in vulnerability to apoptosis compared to cells expressing wild-type tau and to cells expressing the N279K mutation. The cell death-enhancing effect of the V337M mutation was largely abolished when cells were treated with nifedipine, indicating a requirement for calcium influx through L-type. Taxol reduced the vulnerability of cells expressing V337M tau to a level similar to that of nifedipine-treated cells, consistent with a requirement for microtubule depolymerization in the cell death-promoting effect of the mutation. The V337M tau mutation, which reduces its ability to bind microtubules, enhances VDCC activity resulting in increased calcium influx and increased susceptibility of neurons to death. These findings suggest that excessive calcium influx may play a role in the neurodegeneration in FTDP-17. Consistent with this possibility, levels of calcium and calcium-dependent protease activity are increased in neurofibrillary tangles, and elevation of intracellular calcium levels can induce alterations in the cytoskeleton of neurons similar to those seen in neurofibrillary tangles. Functional interactions between microtubules and ion channels are suggested by studies showing that colchicine alters sodium currents in brainstem neurons and enhances calcium influx-dependent neurotoxicity in cultured hippocampal neurons. Microtubule depolymerization was also reported to enhance calcium currents in cardiac myocytes. Our data suggest that the V337M tau mutation exerts a specific effect on VDCC channel function that involves decreased rundown of the channel activity. Stabilization of microtubules with taxol can protect neural cells against calcium-mediated cell death. The ability of taxol to prevent the enhancement of calcium currents by V337M tau suggests that enhanced microtubule depolymerization is involved in the calcium signaling defect caused by the mutation, and further suggests that an abnormality in the microtubule-regulating function of tau results in dysregulation of neuronal calcium homeostasis in FTDP-17.